Glycated protein is nonenzymatically-glycated protein. Specifically, the glycated protein is generated as a result of nonenzymatical covalent bonding between an aldehyde group on the sugar; that is, on the aldose side (a monosaccharide potentially having an aldehyde group and its derivative) and an amino group on the protein side. Examples of such amino group on the protein side include N-terminal α-amino group and side-chain ε-amino group of internal lysine residue. α-glycated protein and/or ε-glycated protein is generated by glycation of these groups. Furthermore, such glycated protein is formed when a Schiff base generated as a reaction intermediate is subjected to Amadori rearrangement. Thus, the glycated protein is also referred to as a so-called Amadori compound.
The glycated protein is contained in body fluids such as in vivo blood or biological samples such as hair. Examples of such glycated protein existing in blood include glycated hemoglobin and glycoalbumin. Generation of such glycated protein strongly depends on the concentrations of saccharides such as glucose, which are dissolved in sera. Under diabetic conditions, it is known that glycated protein generation is enhanced. Furthermore, glycoalbumin or glycated hemoglobin is used as an indicator that reflects a blood glucose level for diagnosing or controlling the symptoms of diabetes. In particular, glycated hemoglobin contained in erythrocytes reflects a past average blood glucose level for a certain time period. Hence, quantitative determination of such glycated protein as an indicator is important for diagnosing or controlling the symptoms of diabetes.
A method that is conventionally known as an enzymatic method for quantitative determination of glycated protein and is intended for use in diagnosing diabetes involves digesting a glycated protein to be determined with protease or the like, liberating glycated peptide or glycated amino acid, and quantitatively determining the subject glycated protein using enzymes that specifically act thereon. Another example of a known method involves determining a glycated amino acid derived from glycated hemoglobin or glycoalbumin using oxidase that acts on the glycated amino acid (see JP Patent Publication (Kokoku) No. 5-33997 B (1993); JP Patent Publication (Kokoku) No. 6-65300 B (1994); JP Patent Publication (Kokai) No. 5-192193 A (1993); JP Patent Publication (Kokai) No. 6-46846 (1994) A; and International Publication No. 97/13872 Pamphlet). Furthermore, a method disclosed by the applicant relates to quantitative determination of hemoglobin A1c (glycated protein in which glucose binds to an α-amino group of hemoglobin “β chain” N-terminal Val (valine), hereinafter referred to as HbA1c), the precise quantitative determination of which has been difficult with the above methods, and uses an enzyme (fructosyl peptide oxidase) that acts on an α-glycated peptide (see JP Patent Publication (Kokai) No. 2001-95598 A).
There are 4 types of glycated amino acid or glycated peptide that can be liberated from a glycated protein in connection with the above various types of determination and can serve as subjects for determination: α-glycated amino acid, α-glycated peptide, ε-glycated amino acid, and ε-glycated peptide. α-glycated amino acids and α-glycated peptides are generated via their liberation from the glycated N-termini of glycated proteins. Meanwhile, an ε-glycated amino acid and an ε-glycated peptide are generated via their liberation from positions where the ε-side chains of amino acids are glycated (for example, glycation can take place at a lysine residue or an arginine residue within the protein).
For example, for quantitative determination of glycoalbumin, the sum of the amount of ε-glycated amino acid and the amount of ε-glycated peptide is measured. For quantitative determination of HbA1c, α-glycated peptide determination is performed, for example. Depending on glycated protein types to be determined, different glycated amino acids and/or glycated peptides are each determined using an enzyme having high specificity thereto. At this time, if the amount or the concentration of a target glycated amino acid or glycated peptide (that is cleaved by protease from a true determination subject) includes an amount or concentration of glycated amino acids or glycated peptides other than the target, a measured value higher than the actual measured value of the target (specific) glycated amino acid or glycated peptide is obtained. Thus, a measurement error is generated. As a countermeasure for such problem, 2 methods relating to quantitative determination of glycated hemoglobin have been disclosed, which involve eliminating glycated amino acids or glycated peptides other than a substance to be determined (see International Publication No. 02/061119 Pamphlet and JP Patent Publication (Kokai) No. 2004-113014 A).
The 1st method involves eliminating in advance a liberated ε-glycated amino acid within a sample using an enzyme with high specificity to the ε-glycated amino acid and then determining an α-glycated amino acid alone with the use of a measurement enzyme that acts on both the α-glycated amino acid and the ε-glycated amino acid (see International Publication No. 02/061119 Pamphlet). The use of the method makes it possible to avoid partial inclusion of the measured value of an ε-glycated amino acid or an ε-glycated peptide liberated in a sample in the measured value of a target substance. However, for example, in the case of patients or the like subjected to infusion of a high-calorie amino acid solution, the method is problematic in that a liberated α-glycated amino acid that can be generated in blood during or after infusion cannot be eliminated. Specifically, such an α-glycated amino acid contaminant cannot be eliminated with the use of an enzyme for elimination. Furthermore, such an enzyme for determination is unable to distinguish between an α-glycated amino acid that has been liberated and an α-glycated amino acid that has been cleaved with protease. Therefore, the value obtained by this method includes the amount of such a liberated α-glycated amino acid that has remained uneliminated. This can cause an error.
The 2nd method involves using an enzyme that acts on an α-glycated amino acid or an α-glycated peptide for both elimination and determination (see JP Patent Publication (Kokai) No. 2004-113014A). Specifically, a substrate on which an enzyme for determination can act is eliminated in advance from a sample before protease treatment, and then only a substrate for determination, which is newly generated by protease treatment, is determined. The α-glycated amino acid or the α-glycated peptide derived from only the subject glycated protein is thus determined. According to this method, a measurement error resulting from a contaminant that is a substrate of an enzyme for determination (=enzyme for elimination) and is in a state of being liberated before protease treatment can be avoided. However, a problem resulting from the substrate specificity of the enzyme for determination (=enzyme for elimination) still remains. With the use of this method, a model contaminant simulating an ε-glycated amino acid is successfully eliminated. Specifically, an enzyme for determination (=enzyme for elimination) in this method can act well on both an α-glycated amino acid or α-glycated peptide to be determined and an ε-glycated amino acid to be eliminated. However, a sample to be subjected to protease treatment contains a mixture of hemoglobin, blood proteins other than hemoglobin contaminated in the sample, and a plurality of proteins including protease itself. Each of these proteins contains a plurality of ε-glycated portions, so that these portions are also liberated by protease treatment, generating ε-glycated amino acids and/or ε-glycated peptides. Hence, inclusion of the measured values of such amino acids and/or peptides in the measured value of a target substance because of an enzyme for determination cannot be avoided. Therefore, it cannot be said that this method is sufficient for precise determination of an α-glycated amino acid or a glycated peptide alone.
As described above, it has been desired for enzymatic determination of a glycated protein to further improve technology relating to a method and a reagent for eliminating a glycated amino acid and/or a glycated peptide (for eliminating substances that cause measurement errors).